Mild Hypothermia Promotes Ischemic Tolerance and Survival of Neural Stem Cell Grafts by Enhancing Global SUMOylation

Department of Neurosurgery, Shengjing Hospital of China Medical University, Shenyang 110004, China Liaoning Clinical Medical Research Center in Nervous System Disease, Shenyang 110004, China Key Laboratory of Neuro-oncology in Liaoning Province, Shenyang 110004, China Central Laboratory, The Fifth Central Hospital of Tianjin, Tianjin 300450, China Tianjin Key Laboratory of Epigenetics for Organ Development of Preterm Infants, The Fifth Central Hospital of Tianjin, Tianjin 300450, China


Introduction
China ranks first in the world for the number of people experiencing stroke and, with the advent of an aging population, this trend is increasing annually [1][2][3]. According to reports, the prevalence of ischemic stroke in China was 1981 per 100,000 in 2017, with a mortality rate of 149 per 100,000 [2], thus imposing a heavy burden on families and society. Current treatment measures for cerebral infarction involve basic support and monitoring, dehydration to reduce intracranial pressure, anticoagulation, scavenging of free radicals, and nourishing nerves in an attempt to prevent complications and reduce mortality [4,5]; however, the efficacy of all these methods remains uncertain. Therefore, in clinical practice, the implementation of an effective treatment plan is particularly important for improving the survival of patients and their quality of life.
In recent years, NSCs have yielded high hopes for the treatment of stroke, especially ischemic cerebrovascular disease [6]. Theoretically, NSCs transplanted into the penumbra at the edge of cerebral infarction will proliferate for a few generations and then differentiate to supplement neurons and glial cells, thereby repairing damage and improving nerve function. However, in fact, the penumbra microenvironment exhibits severe hypoxia and accumulation of large amounts of toxic substances that are extremely unfavorable for the local survival of transplanted NSCs, which severely limits their application. Therefore, improving the survival of transplanted NSCs in the penumbra is key for the treatment of ischemic cerebrovascular disease.
Nowadays, the application of mild hypothermia for brain protection has attracted increasing attention and gradually been implemented in clinical practice. A large number of international trials have confirmed the effectiveness and practicability of mild hypothermia in clinical applications, which can reduce the mortality rate and effectively improve the quality-of-life of patients with ischemic cerebrovascular disease [7][8][9]. However, as most reports only describe the clinical efficacy and methods of mild hypothermia treatment, the exact mechanism of its action has not been clarified. This restricts its wide acceptance by doctors and, to a certain extent, widespread promotion in clinical practice. Therefore, it is necessary to have a deeper and comprehensive understanding of the molecular mechanism by which mild hypothermia protects the brain to help doctors provide more precise treatment plans for patients with cerebral ischemia.
Small ubiquitin-like modifier-(SUMO-) mediated SUMOylation, a form of posttranslational modification of proteins, is used by cells to respond to external stress and adapt to changes in the internal environment [10]. SUMO modification of proteins requires the cascade reaction of SUMO activating enzyme (E1), conjugating enzyme (E2 and UBC9), and ligase enzyme (E3) [11][12][13]. Neurons can reportedly antagonize the adverse microenvironment of hypoxia by increasing global SUMOylation of a large number of proteins, such as hypoxia-inducible factor 1α (HIF-1α), and mild hypothermia can further increase global SUMOylation in neurons [14][15][16]. Indeed, this enriches the molecular mechanism underlying mild hypothermia-induced brain protection to a certain extent. At present, no reports describe the effects of hypoxia and mild hypothermia on protein SUMOylation in neural stem cells (NSCs). Moreover, it is unknown whether transplantation of NSCs overexpressing SUMO into the edge of a cerebral infarction, with or without mild hypothermia, can increase the survival rate of NSC grafts and improve prognosis.
Therefore, this study investigated the effects of hypoxia and mild hypothermia on global SUMOylation of NSCs, as well as their proliferation, differentiation, and hypoxia tolerance. We also transplanted NSCs overexpressing UBC9 into the cerebral ischemic penumbra of a rat middle cerebral artery occlusion (MCAO) model to evaluate their survival in vivo, as well as effects on the neurological functions of rats. In summary, the results show that mild hypothermia can promote the ischemic tolerance and survival of NSC grafts by enhancing global SUMOylation and improve the neurological function of rats. These conclusions identify a molecular mechanism supporting the brain protection elicited by mild hypothermia and provide a guide for increasing the survival of NSC grafts to improve the prognosis of patients with cerebral infarction.

Materials and Methods
The research conforms to NIH (2011) Guide for the Care and Use of Laboratory Animals (8th Edition, Institute for Laboratory Animal Research, Division on Earth and Life Studies, National Research Council of the National Academies Press).

Experimental Rats.
Total number of 60 12-week-old male and 5 new-born (within 1 day) female Sprague-Dawley rats were purchased from SPF Biotechnology Co., Ltd. (Beijing, China). These rats were housed in the Animal Experimental Center of the Fifth Central Hospital of Tianjin (Tianjin, China) with 50% ± 5% humidity and 20-25°C ambient.
2.2. NSC Culture and Treatment. Rat NSCs were isolated and extracted from the hippocampus of new-born rats (within 1 day) under sterile conditions, digested with 0.05% trypsin for 15 minutes, and carefully pipetted with a dropper to form a single cell suspension at 1000 r/min. Trypsin was removed after centrifugation for 5 min. Neural stem cells were cultured in rat neural stem cell culture medium (Cyagen Biosciences, Suzhou, China). After 5-7 days of culture, the neurospheres were dissociated into single cell suspensions by mechanical separation for subculture, and the cells were seeded at a density of 2 × 10 5 cells/. Cells of neurospheres were confirmed to be NSCs and propagated for 2 passages to obtain enough NSCs for experiments. Cultures maintained at 37°C and 5% CO 2 in an incubator were recorded as the control group (Con). Hypoxia was performed by placing NSCs in 1% O 2 , 94% N 2 , 5% CO 2 , balanced nitrogen, and 95% humidity for 12 h (designated as H 12 h). For the mild hypothermia group, incubators were set at 33°C (designated as 33°C). All the experiments were carried out after a subsequent 48 h of culture under normal conditions.

Establishment of Rat MCAO Models.
A total number of 60 adult male Sprague-Dawley rats were randomly divided into 5 groups. During the operation, a small-animal ventilator (Shanghai Yuyan Instruments Co., Ltd., Shanghai, China) was used to maintain animals' respiration, and body temperature was monitored by a rectal temperature control. Making a 1 cm longitudinal incision between rat sternum and mandible, then left common carotid artery was isolated, and we found out the external carotid and internal carotid arteries under a stereo microscope (Olympus Corporation, Tokyo, Japan). Next, we ligated the distal heart end of the external carotid artery and the proximal heart end of the carotid artery, and a modified nylon thread (with 0.23 mm head diameter and 0.18 mm trunk diameter) was inserted from the carotid artery to the middle cerebral artery (~12.0 mm deep) and fixed with surgical line.

Mild Hypothermic Treatment.
Rats which underwent surgery without thread insertion were defined as the "sham operation" group. NSCs or UBC9 NSCs were transplanted to the ischemic penumbra after the establishment of MCAO models as reported in literatures [17,18]. To make mild hypothermia, MCAO rats injected with NSCs were placed on an insulation blanket (Shanghai Yuyan Instruments), and a rectal temperature monitor was to keep body temperature at 32 to 34°C for 12 h. Rats were then removed from blanket and gradually recovered to normal body temperature. After removal, some models' brains were stripped and sliced then incubated in 1% 2,3,5-triphenyl-2H-tetrazolium chloride (TTC, Sigma-Aldrich, St. Louis, MO) for 20 min at 37°C to distinguish the infarct area (white) and the uninfarct area (pink and red). Sections were fixed with 4% paraformaldehyde for 2 h to distinguish stained from unstained areas. The infarcted and uninfarcted areas were analyzed by ImageJ software (National Institutes of Health, Bethesda, MD), and percentages of infarct were calculated as ðinfarct areaÞ/ðarea of the whole brain sliceÞ × 100%. Brain tissues under the same conditions should be collected for paraffin section and immunofluorescence staining assay as described in Section 2.6.
2.9. Neural Function Analysis. Animals recovered from MCAO for 1, 4, 7, 14, 21, or 28 days before assess neurological functions by modified Neurological Severity Scores (mNSS) [19,20]. The mNSS consisted of balance, movement, sensory function, and reflex tests with a score ranging from 0 to 18 (normal score: 0; maximum defect score: 18). In addition, rotarod testing was performed to evaluate of rat neurological deficits (Zhishuduobao Biotechnology Co., Ltd, Beijing, China). Rats were pretrained for 3 times before MCAO, and the experiment was performed for 7 days after MCAO. The cylinder accelerated from 10 to 40 rpm within 5 min; before rats fell from the rod, the latency to fall was recorded. Mean latency for each rat was calculated from three trials with a 30 min interval between 2 trials.
3 Oxidative Medicine and Cellular Longevity 2.10. Rat Behavior Tests. 7 days after MCAO, rats should be tested for behaviors. Spontaneous activity was monitored within 30 min for distance traveled and time spent in corners. Activity was assessed as distance traveled (locomotion), vertical activity (rearing), thigmotaxis, and time spent in corners using behavioral tester (Zhishuduobao Biotechnology Co., Ltd, Beijing, China) equipment. On the 10th day after MCAO, rats were tested for memory and learning abilities in the Morris water maze (Zhishuduobao Biotechnology Co., Ltd, Beijing, China) according to reference [21]. Rats underwent the visible platform experiment for the first 2 days, the nonvisible platform experiment for the following 3 days, and the probe trial for the last day. Escape latency and swimming paths were measured during the first 5 days. In the probe trial, percentages of time spent in quadrant IV and numbers of platform crossing were recorded.
2.11. Data Statistics and Analysis. Each experiment was performed at least for 3 times. Data are showed as mean ± standard deviation (SD) and were analyzed using GraphPad  Oxidative Medicine and Cellular Longevity Prism 6 software (San Diego, CA). A P value <0.05 was considered significant difference. One-way ANOVA or the unpaired Student's t test was used to evaluate the significance of differences among treatment groups, as appropriate.

Results
3.1. Hypoxia Increased Injury, Inhibited Differentiation, Increased the Stemness Maintenance Potential, and Reduced the Metabolic Capacity of NSCs. After 12 h of hypoxia stimulation, the content of LDH released by NSCs increased significantly, suggesting cell damage (Figure 1(a)). The results showed that the percentage of apoptotic cells reached 30% after hypoxia (Figure 1(b)). Immunofluorescence detection showed that a certain proportion of NSCs spontaneously differentiated under normal conditions. NSE and GFAP were expressed. Nestin, a marker of NSCs, was highly expressed, and cells showed extensional growth morphology similar to nerve fiber structures. After hypoxia, NSCs exhibited obvious spherical growth, NSE and GFAP expression decreased, and nestin expression significantly increased (Figure 1(c)). Moreover, expression of stem cell markers Oct4 and SOX2 increased significantly (Figure 1(d)). Additionally, we calculated ECAR levels and found that hypoxia could significantly increase the anaerobic hydrolysis level of NSCs (Figure 1(e)).   (Figure 2(c)). Expression of Oct4 and SOX2 was also increased by mild hypothermia compared with cells at 37°C exposed to hypoxia (Figure 2(d)). Mild hypothermia could reduce the metabolic level of cells and inhibit hypoxiainduced increases of anaerobic fermentation (Figure 2(e)).

Hypoxia Increased Whole-Protein SUMOylation in NSCs and Mild Hypothermia further Strengthened SUMOylation
Modification. Western blot was used to detect whole-cell levels of the SUMOylation modification under the conditions of hypoxia, mild hypothermia, and their superposition. The results showed that hypoxia and hypothermia could significantly promote the binding of SUMO1 and SUMO2/3 to target proteins and had a superposition effect; however, it had little effect on free SUMOs. Further detection of conju-gating enzyme E2 (UBC9) showed that hypoxia could promote the expression of this protein (Figures 3(a)-3(c)).

Overexpression of UBC9 Could Increase the Stemness and
Hypoxia Tolerance of NSCs. We transfected NSCs with a plasmid carrying the UBC9 gene sequence and screened clones with high expression of UBC9. Protein detection showed that SUMO1 and SUMO2/3 conjugates in UBC9overexpressing NSCs were significantly increased, as were the contents of stemness maintenance molecules Oct4 and SOX2 (Figures 4(a)-4(c)). Immunofluorescence detection showed that UBC9 overexpression could significantly increase the expression of the NSC marker nestin and promote spherical growth of cells. Under hypoxia, expression of differentiation markers in NSCs was further reduced, nestin expression was increased, and the cell ball became smaller and round (Figure 4(d) H 12h=33˚C * * * * * * * * * * * * * * * * * (c) Figure 3: Effects of hypoxia (H 12 h) and mild hypothermia (33°C) on whole-protein SUMO modification in NSCs. (a) and (b) Expression of SUMO1 and SUMO2/3 conjugates, free SUMO1 and SUMO2/3, and UBC9 in NSCs after hypoxia and/or moderate hypothermic treatment, as assessed by western blotting. (c) Quantitative data were normalized to GAPDH and are expressed as mean ± SD (n = 3). * P < 0:05, * * P < 0:01, and * * * P < 0:001 vs. control. 6 Oxidative Medicine and Cellular Longevity damage after hypoxia, but there was no significant damage to cells under normoxia (Figure 4(e)). Hypoxia activated the cleaved caspase-3 apoptosis signal in NSCs and pro-moted apoptosis, but UBC9 overexpression could partially reverse the proapoptotic damage induced by hypoxia (Figure 4(f)).  7 Oxidative Medicine and Cellular Longevity 3.5. siUBC9 Reduced the Stemness and Hypoxia Tolerance of NSCs. We used small interfering RNA sequences to knockdown UBC9 expression. Protein detection showed that low UBC9 expression inhibited the modification of target proteins by SUMO1 and SUMO2/3 in NSCs, and expression of the stemness maintenance molecules Oct4 and SOX2 was significantly decreased (Figures 5(a)-5(c)). Immunofluorescence detection showed that low UBC9 expression could significantly inhibit nestin expression and promote cell differentiation. However, under hypoxic conditions, expression   Oxidative Medicine and Cellular Longevity increased and promoted the expression of its precursor form ( Figure 5(f)). Thus, low UBC9 expression significantly aggravated the hypoxic injury of NSCs.

Mild Hypothermia Increased the Survival of NSCs
Transplanted into the Cerebral Ischemic Penumbra of Mice and Improved Neuromotor Function. MCAO could generate infarct areas reaching 50% in rats, and the infarct area could be reduced to 30% after transplantation of NSCs into the penumbra, while mild hypothermia could further reduce the infarct area. Immunofluorescence staining showed that NSC transplantation could significantly increase the density of NSCs in the penumbra, and mild hypothermia could further increase the survival of NSCs (Figures 6(a) and 6(b)). Compared with the MCAO group, the motor and coordination ability of rats transplanted with moderately hypother-mic NSCs improved after 7 days (Figure 6(c)), and the neural function of rats significantly improved after 21 days (Table 1). Notably, postoperative MCAO rats showed anxiety, which was significantly relieved after NSC transplantation and mild hypothermia (Figures 6(d) and 6(e)). Morris water maze testing showed that the learning and memory function of rats transplanted with NSCs into the penumbra and treated with mild hypothermia improved to varying degrees compared with after MCAO (Figures 6(f)  days following surgery in each group. Data are expressed as mean ± SD (n = 7). # P < 0:05 vs. MCAO and & P < 0:05 vs. MCAO+NSC.

Transplanted NSCs Overexpressing UBC9 in the Cerebral
Ischemic Penumbra of Rats Exhibited Higher Survival Rates and Enhanced Neuromotor Function. Compared with simple transplantation of NSCs, UBC9-overepxressing NSCS could reduce the cerebral infarction area of rats from 30% to 12%. Moreover, overexpression of UBC9 could promote the survival of NSCs, as well as their ability to adapt to hypoxic injury of brain (Figures 7(a) and 7(b)). Compared with the NSC-transplanted group, MCAO rats transplanted with NSCs overexpressing UBC9 exhibited significantly improved motor ability and neurological functions (Table 1, Figure 7(c)), reduced anxiety levels, and enhanced learning functions to varying degrees (Figures 7(d)-7(h)).

Discussion
In this study, we first examined the effect of hypoxia on NSCs. The results show that although hypoxia damaged NSCs, it increased their potential to maintain stemness, inhibited their neuronal differentiation, and reduced their metabolism. Further studies showed that mild hypothermia antagonized hypoxia-induced damage to NSCs, further inhibiting their differentiation and reducing cell metabolism. To evaluate whether the protective effect of mild hypothermia on NSCs was related to the SUMO modification of proteins, we examined the effect of mild hypothermia on the expression of SUMOs in NSCs. The results show that hypoxia increased global SUMO modifications of NSCs, both SUMO1 and SUMO2/3, and mild hypothermia further strengthened these trends. These results preliminarily validated our hypothesis that hypoxia can increase the hypoxia tolerance of NSCs by increasing global SUMOylation, and mild hypothermia can enhance neuroprotective effects by reinforcing this trend.
To evaluate whether global SUMOylation is indispensable to improving the hypoxia tolerance of NSCs, we next overexpressed and silenced the UBC9 gene (the only E2binding enzyme in the SUMO modification reaction) [22] in NSCs and evaluated their hypoxia tolerance and stemness maintenance potential. The results show that Ubc9 overexpression increased both the stemness potential of NSCs and their tolerance to hypoxia; in contrast, silencing UBC9 reduced the stemness of NSCs and decreased their tolerance to hypoxia. These results indicate that global SUMO modification of target proteins is essential to improve the tolerance of NSCs to hypoxia. However, we cannot specify exactly which proteins were SUMO-modified during this event. According to previous studies, we speculate that hypoxia greatly increases SUMO modification levels of numerous target proteins, including HIF-1α, Oct4, and SOX2. Theoretically, SUMO-modified HIF-1α, Oct4, and SOX2 cannot be degraded by ubiquitin hydrolase [23][24][25], thus allowing these proteins to persist and stably exist in the nucleus and cytoplasm of NSCs, whereby they increase hypoxia tolerance and stemness potential.
Finally, we established an MCAO model in rats to investigate the effects of mild hypothermia on survival of NSCs transplanted in the cerebral ischemic penumbra and subsequent improvements of neurological function. The results show that mild hypothermia increased the survival of NSCs transplanted in the cerebral ischemic penumbra of rats and improved their neurological functions, including motor and learning abilities. Finally, we transplanted NSCs overexpressing UBC9 into the cerebral ischemic penumbra of rats and found that these cells exhibited higher survival rates and better improved the neurological function of experimental animals, including motor and learning functions.
These results verify our hypothesis that increasing global SUMOylation of many target proteins, e.g., by overexpressing UBC9, can help NSCs obtain stronger hypoxia tolerance. Moreover, transplantation of NSCs with stronger hypoxia tolerance into the cerebral ischemic penumbra and subsequent mild hypothermia treatment increased the survival of NSCs in the penumbra and enhanced the neurological function of animals.
Although this study has enriched new ideas for the future treatment of ischemic cerebrovascular disease with mild hypothermia therapy combined with NSCs transplantation, there are still many difficult problems to be solved before it is actually applied to the clinic. First, due to the existence of the blood-brain barrier, how to inoculate NSCs into the brain of patients with cerebral ischemia will be a difficult problem for clinicians to face. Second, how to control the time window of hypothermia cooling and rewarming to produce the best therapeutic effect on patients is far from being interpreted. Third, the potential tumorigenic effects brought about by the proliferative characteristics of NSCs are serious side effects that we can never ignore. Last but not the final, SUMOylation can simultaneously intervene in the posttranslational modification of thousands of proteins. How to achieve advantages and avoid disadvantages at the molecular level and protein function will be a problem that researchers attach great importance to. In conclusion, there is still a long way to go before the clinical application of mild hypothermia combined with NSCs transplantation in the treatment of ischemic cerebrovascular disease.

Conclusions
Global protein SUMO modification is an important molecular mechanism for NSC tolerance of hypoxia, and mild hypothermia can further increase the degree of global protein SUMO modification in NSCs-a newly discovered molecular mechanism by which mild hypothermia protects the brain. Indeed, mild hypothermia can improve the hypoxia tolerance of NSCs and increase their survival following transplantation in situ, thus yielding better nerve repair effects.

Data Availability
All the data used to support the findings of this study are available from the corresponding authors upon request.

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